Bottom Line:
In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

ABSTRACTBacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

pone-0014682-g003: Dynamics of Bin-induced vacuolation.(A) Bin-treated (for 24 h) subconfluent MDCK-Cpm1 cells were submitted to time-lapse phase contrast video microscopy, as exemplified in video S1. At the indicated period of time the percentage of MDCK-Cpm1 cells displaying at least one vacuole was determined. Mean values ± SD, n = 3. In the same videos, the percentage of cells that present post-mitotic vacuolation among the cells that display at least one vacuole, as illustrated in (B) was determined. Mean values ± SD, n = 3. (B) Bin-treated cells displaying the post-mitotic vacuolation phenomenon; images selected from video S2. The selection starts 15h after intoxication, intervals in hours. Black and white arrows pinpoint two dividing cells. Bars, 10 µm.

Mentions:
In order to determine the dynamics of Bin-induced vacuolation of MDCK-Cpm1 cells we proceeded to time-lapse video microscopy. Vacuoles appeared within the first hour of intoxication in a subset of the cell population and rapidly increased in a time-dependent manner, finally affecting more than 50% of the cell population (Fig. 3A and Video S1). Strikingly, this vacuolation was transient, as the number of vacuolated cells started to decrease after 6 h of intoxication. Interestingly, at late time points of intoxication the vacuolation still affected about 25% of the cell population (Fig. 3A). By looking at individual cells we found that vacuolation could be reversed within a few hours. However, as shown in video S1 and S2 and figure 3B, we observed that the vacuolation reappeared in dividing cells and was dramatic after cytokinesis. We named this newly described phenomenon post-mitotic vacuolation. In fact, the number of cells vacuolating after mitosis increased as a function of time, and 24 h after intoxication almost all of the vacuoles were detected in post mitotic cells (Fig. 3 A). These results suggest that Bin-induced vacuolation is a transitory phenomenon that is recurrent in newly-divided cells.

pone-0014682-g003: Dynamics of Bin-induced vacuolation.(A) Bin-treated (for 24 h) subconfluent MDCK-Cpm1 cells were submitted to time-lapse phase contrast video microscopy, as exemplified in video S1. At the indicated period of time the percentage of MDCK-Cpm1 cells displaying at least one vacuole was determined. Mean values ± SD, n = 3. In the same videos, the percentage of cells that present post-mitotic vacuolation among the cells that display at least one vacuole, as illustrated in (B) was determined. Mean values ± SD, n = 3. (B) Bin-treated cells displaying the post-mitotic vacuolation phenomenon; images selected from video S2. The selection starts 15h after intoxication, intervals in hours. Black and white arrows pinpoint two dividing cells. Bars, 10 µm.

Mentions:
In order to determine the dynamics of Bin-induced vacuolation of MDCK-Cpm1 cells we proceeded to time-lapse video microscopy. Vacuoles appeared within the first hour of intoxication in a subset of the cell population and rapidly increased in a time-dependent manner, finally affecting more than 50% of the cell population (Fig. 3A and Video S1). Strikingly, this vacuolation was transient, as the number of vacuolated cells started to decrease after 6 h of intoxication. Interestingly, at late time points of intoxication the vacuolation still affected about 25% of the cell population (Fig. 3A). By looking at individual cells we found that vacuolation could be reversed within a few hours. However, as shown in video S1 and S2 and figure 3B, we observed that the vacuolation reappeared in dividing cells and was dramatic after cytokinesis. We named this newly described phenomenon post-mitotic vacuolation. In fact, the number of cells vacuolating after mitosis increased as a function of time, and 24 h after intoxication almost all of the vacuoles were detected in post mitotic cells (Fig. 3 A). These results suggest that Bin-induced vacuolation is a transitory phenomenon that is recurrent in newly-divided cells.

Bottom Line:
In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

ABSTRACTBacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.